Preparation method for nucleic acid sample derived from skin stratum corneum

Abstract

A method for preparing a nucleic acid sample derived from skin stratum corneum includes: extracting nucleic acid from stratum corneum collected from a skin surface to prepare a water-soluble alcohol aqueous solution containing the nucleic acid; contacting the nucleic acid in the water-soluble alcohol aqueous solution with a solid-phase material to separate the nucleic acid; and amplifying, or reverse-transcribing and amplifying, the separated nucleic acid in the presence of single-stranded nucleic acid-binding protein to prepare a nucleic acid sample.

1 . A method for preparing a nucleic acid sample derived from skin stratum corneum, the method comprising:
 extracting RNA from stratum corneum collected from a skin surface to prepare an ethanol aqueous solution containing the RNA, wherein the stratum corneum is stratum corneum within a depth of 5 μm from the skin surface;   contacting the RNA in the water-soluble alcohol aqueous solution with a silica-based solid-phase material to separate the RNA, wherein a concentration of the ethanol in the ethanol aqueous solution is from 40 to 46 vol % before the contact with the silica-based solid-phase material; and   reverse-transcribing and amplifying, the separated RNA in the presence of single-stranded nucleic acid-binding protein to prepare a nucleic acid sample, wherein the amplification is amplification by multiplex PCR, and the single-stranded nucleic acid-binding protein is T4GP32 or a variant thereof.   
     
     
         2 . The method according to  claim 1 , further comprising collecting stratum corneum from the skin surface. 
     
     
         3 . The method according to  claim 1 , wherein the stratum corneum collected from the skin surface is stratum corneum within four stratum corneum layers from the skin surface. 
     
     
         4 . The method according to  claim 2 , wherein the collection of stratum corneum from a skin surface is collection of stratum corneum by tape stripping. 
     
     
         5 . The method according to  claim 4 , wherein adhesive tape used in the tape stripping is acrylic adhesive tape. 
     
     
         6 .- 7 . (canceled) 
     
     
         8 . The method according to  claim 1 , wherein the reverse transcription and amplification are multiplex RT-PCR, and an initial concentration of the single-stranded nucleic acid-binding protein in a reaction solution for the multiplex RT-PCR is from 3 to 300 μg/mL. 
     
     
         9 .- 11 . (canceled) 
     
     
         12 . The method according to  claim 1 , wherein the T4GP32 or a variant thereof is protein that consists of an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having a sequence identity of at least 90% with the sequence and binds to single-stranded nucleic acid. 
     
     
         13 . The method according to  claim 1 , wherein the silica-based solid-phase material is a solid-phase material including a porous silica membrane. 
     
     
         14 . A method for detecting gene expression in skin, the method comprising detecting gene expression in the nucleic acid sample derived from skin stratum corneum prepared by the method according to  claim 1 . 
     
     
         15 . The method according to  claim 1 , wherein the concentration of the ethanol in the ethanol aqueous solution is from 42 to 45 vol %. 
     
     
         16 . The method according to  claim 14 , wherein the gene expression is detected by sequencing.